Phellinus linteus (Berk. and Curt.) Teng, Hymenochaetaceae (PL), a type of medicinal mushroom, were examined in rats subjected to myocardial IR injury. The left main coronary artery of rats was ligated for 1 h and reperfused for 3 h. The arrhythmia levels were monitored during the entire process and the infarct size was evaluated after myocardial IR injury. Furthermore, the expression levels of proteins in apoptotic and autophagic pathways were observed. Pretreatment with PL mycelium (PLM) significantly reduced ventricular arrhythmia and mortality due to myocardial IR injury. PLM also significantly decreased myocardial infarct size and plasma lactate dehydrogenase level after myocardial IR injury. Moreover, PLM administration resulted in decreased caspase 3 and caspase 9 activation and increased Bcl-2/Bax ratio. Phosphorylation level of AMPK was elevated while mTOR level was reduced. Becline-1 and p62 levels decreased. These findings suggest that PLM is effective in protecting the myocardium against IR injury. The mechanism involves mediation through suppressed pro-apoptotic signaling and regulation of autophagic signaling, including stimulation of AMPK-dependent pathway and inhibition of beclin-1-dependent pathway, resulting in enhancement of protective autophagy and inhibition of excessive autophagy.The incidence of myocardial ischemia-reperfusion (IR) injury is rapidly increasing around the world and this disease is a major contributor to global morbidity and mortality. It is known that regulation of programmed cell death including apoptosis and autophagy reduces the impact of myocardial IR injury. In this study, the cardioprotective effects and underlying mechanisms of  Myocardial ischemia is caused by a lack of coronary blood supply to the heart. During myocardial ischemia, a characteristic pattern of metabolic and ultrastructural changes leads to irreversible injury. To limit myocardial infarct size and reduce mortality, early restoration of blood flow to the ischemic myocardium is a common treatment strategy . HoweverRecent studies have shown that myocardial cell death is the main reason for the poor prognosis of patients with ischemic heart disease. Ischemic and reperfusion injuries to the heart cause various types of cardiomyocyte death such as necrosis, apoptosis, and autophagy . Among tIt has been reported that crosstalk exists between apoptosis and autophagy . ActivatPhellinus linteus (Berk. and Curt.) Teng, Hymenochaetaceae (PL), a well-established medicinal mushroom, has been used in Asian countries for centuries to prevent or treat ailments as diverse as hemorrhage, rheumatoid arthritis, gastroenteric dysfunction, diarrhea, and cancers. A number of studies have revealed that PL alleviates septic shock and has antitumor  and ventricular fibrillation (VF), as well as infarct size and mortality. We also investigated the cardioprotective effects of PLM on signaling pathways involved in autophagy. In the present study, the aim was to explore the potential of PLM for protecting against myocardial IR injury in vivo, as well as the underlying mechanisms.ntitumor , immunomntitumor , anti-anntitumor , and antntitumor , 2015. Mntitumor . From thntitumor . There had libitum access to food and water.Sprague-Dawley rats , each weighing 250–300 g, were used. In this study, based on the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The animal study protocol was approved by the Institutional Animal Ethics Committee of Taipei Veterans General Hospital, Taipei, Taiwan (IACUA 2015-241). Animals were housed under conditions of controlled temperature (24 ± 1°C) and humidity (55 ± 5%), as well as 12-h light–dark cycle. Rats were allowed -8 or 10-9 g/kg via jugular vein 15 min prior to left anterior descending artery (LAD) ligation under urethane anesthesia as previously described. The sham and control animals were treated with normal saline. LAD was ligated for 1 h and then reperfused for 3 h. At the end of the experiment, rat hearts were harvested to evaluate infarct size and protein expression levels by triphenyl tetrazolium chloride (TTC) staining and immunoblotting.The rats were randomly assigned to three groups: (1) sham; (2) myocardial IR (control); (3) PLM treatment (PLM). The experimental animals were administered PLM at a dosage of 10Phellinus linteus mycelium was supplied by Chiu-Yeh WU’s lab  and prepared as previously described  slants and incubated at 30°C for 14 days and then stored at 4°C.escribed . P. lint2 of the agar plate culture with the sterilized cutter. The inoculum culture was incubated in a 300 ml baffled flask containing 100 ml potato dextrose broth  under 30°C, 100 rpm on a rotary shaker incubator for 24 h. The production culture in a stirred-tank bioreactor  for PL was carried out on the fermentation medium compositions (w/v): dextrose 2%, peptone 0.1% and malt extract 2%, and was sterilized at 121°C for 30 min. Fermentations were carried out with 3 L fermentation medium under the following conditions: inoculum ratio 5%, temperature 30°C, aeration rate 1.5 vvm and agitation rate 125 rpm. The cultivation was maintained for 20 days after the inoculum was transferred to the medium. After finishing cultivation, the harvest of mycelial biomass were filtered by filter paper, and then the resulting precipitate was washed repeatedly by ddH2O and dried at 50°C to a constant weight. 50 g dried mycelia biomass was extracted by 500 mL ddH2O at 121°C for 15 min, cooled down, and filtered, and this step was repeated twice. The filtrate was collected and condensed by lyophilized. The powder was dissolved and dilute to working concentration (10-8 and 10-9 g/kg) with normal saline.To prepare the experimental inoculant, the microorganism was initially grown on PDA plates, and then transferred to the inoculum medium by punching out six pieces of 1 cmTemporary occlusion of the left main coronary artery was carried out to induce myocardial IR injury, as previously described . BrieflyA respirator for small rodents  was used for ventilation with room air. Stroke volume was set at 10 ml/kg body weight and stroke rate at 60 strokes/min. Following sectioning of the fourth and fifth ribs, at 2 mm to the left of the sternum, left thoracotomy was carried out to open up the chest. Subsequently, the heart was quickly externalized and inverted. Following placement of 6/0 silk ligature around the left main coronary artery, The animal was allowed 15 min of recovery following repositioning of the heart in the chest. If there was arrhythmia or sustained decrease in BP to less than 70 mmHg following the procedure, the animal was excluded from the study.Antiarrhythmic effects of PLM during myocardial IR injury were analyzed. Changes in HR, BP, and ECG were simultaneously recorded on a personal computer with waveform analysis software  before and during ischemia and reperfusion. Ventricular ectopic activity was evaluated based on Lambeth Convention-recommended diagnostic criteria. Incidences and durations of VT and fibrillation (VF) were determined in both surviving rats and in those that eventually died. The duration of VF was recorded until BP was lower than 15 mmHg in rats that died due to irreversible VF.Rats were sacrificed at 4 h post-IR injury for infarct volume analysis (TTC staining) . FollowiLactate dehydrogenase (LDH) activity in plasma was measured to evaluate myocardial cellular damage. Following IR injury, arterial blood was collected from the carotid catheter and mixed with heparin (about 100 IU). LDH activity was determined based on a previously described method . FollowiLeft ventricle samples were homogenized with tissue protein extraction reagent  containing Protease Inhibitor Cocktail . Concentrations of proteins were quantified using a protein assay dye reagent  and bovine serum albumin (BSA) as the standard. Following the mixing with equal volumes of loading buffer , samples were heated to 95°C for 10 min. They were then separated with SDS-PAGE gel and transferred onto PVDF membranes . Blocking of membranes was achieved with 1% polyvinylpyrrolidone (PVP) and 0.05% tween 20 in phosphate buffer solution (PBS) at room temperature for 1.5 h, followed by incubation with primary antibodies including anti-Bcl2 , anti-Bax , anti-caspase 3, anti-caspase 8, anti-caspase 9, and anti-AMPKα and phospho -AMPKα, anti-phosphoinositide 3 kinase (PI3K) p85, anti-phospho PI3K p85, anti-phospho AKT . In addition, anti-mTOR, anti-AKT  and phospho -mTOR ; anti-LC3 ; anti-beclin-1 ; anti-p62  and anti-β-actin  at 4°C overnight. The membranes were washed three times with PBS containing 0.1% tween-20 (PBST) followed by horse radish peroxidase-conjugated secondary antibodies  at 37°C for 1 h. The blots were analzyed with enhanced chemiluminescent system following additional PBST washes.t-test. The differences in incidences of arrhythmia and mortality between IR control and PLM-treated groups were analyzed on Chi-square assay. Statistical significance was defined as p < 0.05. All tests were two-tailed.Data are expressed as mean ± standard error of mean (SEM). Differences in IR-induced cardiac infarction and immunoblotting were analyzed. The differences between IR control and PLM-treated groups were ascertained by one-way analysis of variance. The variables were subsequently analyzed to determine if there were significant differences between the groups using Student’s Figures 1A,B).Fifteen minutes after PLM administration via jugular vein injection there were no significant alterations in mean arterial BP or HR of anesthetized rats. In addition, there were no significant hemodynamic changes between vehicle and PLM-treated groups during 1-h myocardial ischemia and 3-h reperfusion periods  treated groups by 4.5 ± 1.6 and 10.8 ± 3.8 s, respectively. The incidence % of VF was 75% in vehicle-treated animals with dose dependent decreases in PLM-treated groups . Administration of PLM also significantly improved the duration of VF. Duration of VF was 41.8 ± 14.8 s in vehicle group, and significantly decreased to 0.5 ± 0.2 s in 10-8 g/kg PLM-treated group. VF is the severe type of arrhythmia and it is followed by irreversible death. This phenomenon may imply that PLM decreases myocardial IR-induced arrhythmia and mortality in a dose-dependent manner.Effects of PLM on anesthetized rats following myocardial IR-elicited arrhythmia are presented in Figure 2. Evans blue colored remote myocardium blue but not the AAR and infarct area, which are shown in white on TTC analysis . There are no significant difference among the vehicle, 10-9 and 10-8g/kg PLM. AAR/ left vetrcle  demonstrated that the IR model is consistent in our study . Infarct size/AAR % was reduced in PLM 10-8 g/kg-treated group  . The acivity of plasma LDH, a marker of tissue damage, was significant decreased in PLM group (5489 ± 3389 vs. 1718 ± 182 mU/ml) . Hence, administration of PLM decreased the impact of myocardial IR injury.Changes in myocardial infarct size in control rats and rats administered PLM are shown in Figure 3A). Results demonstrated that PLM treatment increases protein expression of Bcl-2, significantly reduces Bax expression and increases ratio of Bcl-2 to Bax in the myocardium after myocardial IR injury when compared with control.Apoptotic cell death often occurs in cardiomyocytes after myocardial IR injury. We investigated the expressions of Bcl-2 family proteins and caspase 3, 9, and 8 to assess the anti-apoptotic effects of PLM in rats subjected to myocardial IR injury . Both the pro-form and active form (cleaved form) of caspase 3 were reduced in PLM-treated group when compared with control. In addition, PLM treatment significantly reduced the ratio of active form to pro-form of caspase 3 when compared with control. In PLM treated group, it was also shown the ratio of active form of caspase 9 was decreased, but active form of caspase 8 did not change significantly . These results indicated that PLM attenuates apoptotic level via intrinsic pathway in IR-injured heart.Caspase 3 is a key protein in apoptotic cells, both in extrinsic and intrinsic pathways. Thus, we used it to quantify the apoptotic level in the heart after IR injury . Immunoblotting revealed that treatment with PLM significantly increases the ratio of p-AMPK to AMPK expression and reduces the ratio of p-mTOR to mTOR expression in IR injured myocardium when compared with control.To examine the role of autophagy in the cardioprotective effect of PLM, we investigated the phosphorylation levels of AMPK and mTOR (p-AMPK and p-mTOR) in left heart ventricle . However, beclin-1, a major protein in the nucleation step of autophagy, and p62, a protein targeting specific cargo of autophagy . Beclin-1 autophagy induction pathway is regarded as deleterious during IR injury (P < 0.05). These findings suggested that PLM regulates autophagy through stimulation of AMPK-dependent mechanism and inhibition of beclin-1-dependent mechanism in rats after myocardial IR injury.LC3 is widely used to monitor autophagy. LC3-I is the cytosolic form. LC3-II is conjugated with phosphatidylethanolamine (PE) and is present in isolation membranes, autophagosomes, and to a much lesser degree in autolysosomes. In our experiments, when compared with control group, LC3-I did not change in the PLM-treated group. The expression of LC3-II and the ratio of LC3-II to LC3-I increased in PLM-treated group, but this increase did not reach significance (utophagy , signifiR injury , and canFigure 5 summarized the effect of PLM in this study. In the ischemic border zone, the leakage of potassium increased extracellular potassium and caused depolarization of myocytes. This depolarization contributes to electrical heterogeneity of conduction that provide a substrate for reentry, bring about polymorphic VT and/or VF (-8 g/kg, during 1-h myocardial ischemia and 3-h reperfusion period. In addition, myocardial infarction was significantly reduced in PLM-treated group. Compared with control group, LDH activity, which serves as an indicator of cellular damage in plasma, significantly decreased in PLM-treated group during myocardial IR injury, which is consistent with the observation of decreased myocardial infarction. The results of this study support the potential application of PLM as a cardioprotective agent in myocardial IR injury.This is the first study to examine the cardioprotective effects of PLM in rats subjected to myocardial IR injury. PLM did not alter the mean arterial BP or HR in studied rats. nd/or VF . AdminisApoptosis, as well as necrosis, often occurs in cardiomyocytes after myocardial IR injury. In the myocardial ischemic core, which is completely deprived of oxygen and nutrients, myocardium dies due to necrosis. However, in the ischemic penumbra, where there is hypoxia after myocardial IR injury, reperfusion accelerates the process of apoptosis compared with permanent occlusion and myocardial cell death occurs via both necrosis and apoptosis . In thisSeveral studies have reported that myocardial cell death during IR injury occurs by apoptosis, by necrosis and in association with autophagy . AutophaIn others researches, it was reported that mTOR manipulate the autophagy was not only regulated by AMPK but also by PI3K-AKT pathway . In physSeveral studies have demonstrated that Bcl-2, an antiapoptotic protein, inhibits beclin-1 via direct binding. This indicates that there is negative regulation of beclin-1-mediated autophagy by Bcl-2, and crosstalk between autophagy and apoptosis . In thisIn this study, we administrate the PLM, not the fruiting body, before myocardial IR injury. In mycelia of PL, the content of protein, amino acid, total sugar, reducing sugar, polysaccharide and saturated fatty acid were more abundant as compared with the fruiting body . There aOur results indicated that PLM attenuates myocardial IR injury. The cardioprotective effect of PLM may be mediated via inhibition of apoptosis and regulation of autophagy, stimulation of AMPK-dependent mechanism and inhibition of beclin-1-dependent mechanism in rats subjected to myocardial IR injury. The findings of this study suggest that the administration of PLM prior to myocardial IR injury is a potential approach for the treatment of IR injury.P. linteus mycelium; H-HS, J-CY, and S-SH wrote the paper. All authors read and approved the final manuscript.H-HS and S-SH designed research; H-HS, Y-CC, J-ML, and Y-HW conducted research and analyzed data; C-YW and C-YT prepared the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MicroRNAs (miRNAs) are important regulators of biology and disease. Recent animal efficacy studies validate the therapeutic benefit of miRNA modulation and underscore the therapeutic value of miRNA-targeting oligonucleotides. However, whether disease conditions (stress) influence the pharmacological effects of an anti-miR is currently unknown. To study the effect of disease on target regulation after anti-miR treatment, we injected animals with anti-miR-208a, a synthetic oligonucleotide that inhibits the cardiomyocyte-specific miR-208a. Our data indicate that the presence of stress increases the number of regulated miR-208a targets, and that higher stress levels correlate with stronger target derepression. Additionally, the type of stress also influences which targets are regulated upon miR-208a inhibition. Studies in a large animal model indicate a similar stress-dependent anti-miR effect. Subsequent in vitro studies suggest that the influence of stress on anti-miR efficacy depends at least in part on increased cellular anti-miR uptake. These data indicate that the pharmacological effect of anti-miRs is stronger under disease conditions, and that both the type and severity of disease determine the therapeutic outcome. These facts will be important for assessing the therapeutic dose and predicting the therapeutic outcome when applying anti-miRs in a clinical setting. Molecular Therapy, Eding et al. show that the efficacy of anti-miR-208a is dependent on the presence of stress. This is partially explained by an increase in cellular uptake of the anti-miR under disease conditions.MicroRNA inhibitors, known as anti-miRs, can function to target disease-related microRNAs. In this issue of  MicroRNAs (miRNAs) are short, single-stranded RNAs that anneal with complementary sequences in target mRNAs, thereby suppressing protein formation. The function of a given miRNA is determined by its mRNA targets.An intriguing feature of miRNA biology has been the minimal effects of miRNA loss of function under homeostatic conditions.Using miR-208a as a model system, we show that stress changes the effect of anti-miR treatment on mRNA targets. In vitro analysis indicates that the influence of stress on anti-miR efficacy might depend on an increase in cellular anti-miR uptake under stress conditions. Together our data show that anti-miRs have stronger pharmacological effects during disease and that the origin of disease determines the therapeutic outcome. These considerations will be important for assessing the therapeutic dose and predicting the therapeutic effect in patients.AntimiRs function through the inhibition of a specific miRNA, and thereby have a derepressive effect on the direct targets of this miRNA. In an effort to explore whether the effect of anti-miR-208a changes under disease conditions, we performed microarray analysis on cardiac tissue from rats treated with either anti-miR-208a or control, and searched for mRNAs with a binding site for miR-208 that were significantly upregulated in the anti-miR-208a-treated group. This was done on left ventricular tissue in rats 8 weeks after they were subjected to sham surgery (sham) or myocardial infarction (MI) (n = 4 per group). Microarray analysis in the sham rats indicated that anti-miR-208a treatment resulted in the derepression of 108 genes that contained a potential miR-208a binding site in their 3′ untranslated region compared with control. However, while using the same treatment regimen, in the MI group inhibition of miR-208a resulted in the derepression of 325 genes that contain a potential miR-208a binding site in their 3′ untranslated region. Although 64 of these anti-miR-208a-regulated targets were overlapping in both the sham- and MI-operated rats, a large portion also appeared to be either sham- or MI-specific .Figure 1To explore these data in more detail, we set out to determine the cardiac level of miR-208a in response to stress and after anti-miR-208a treatment. The levels of miR-208a did not change significantly in response to stress . CompareBased on our microarray and the real-time PCR data, there appeared to be a trend toward a stronger derepression after MI compared with sham A and 1D.Together, these data indicate that treatment with anti-miR-208a leads to derepression of more miR-208a targets after MI than after sham surgery and that the level of derepression is increased during disease.To determine whether the type of stress also influences the targets that are regulated by an miRNA, we compared the effect of anti-miR-208a during cardiac remodeling in response to MI with the effect of anti-miR-208a in Dahl salt-sensitive rats on a high-salt diet.Real-time PCR analysis on cardiac tissue after anti-miR-208a treatment showed a profound inhibition of miR-208a for both MI and Dahl hearts C. Of allThe random selection of overlapping targets showed a strong derepression after anti-miR-208a compared with control A and S5BThese data show that a large portion of targets is consistently regulated across different disease models upon anti-miR-208a treatment. However, the fact that we can observe derepression for different genes depending on the cause of disease implies that miR-208a also regulates a divergent set of gene targets dependent on the disease etiology.Nppa and Myh7, both well-known markers for cardiac stress,Next, we aimed to investigate whether the severity of stress also influences the level of target derepression in response to anti-miR treatment. To this end, we collected tissue from sham hearts and both the remote and the infarct region after MI. Because an infarct induces a local injury to cardiac tissue, the area surrounding the damaged area presumably experiences more stress than more remote tissue. Indeed, the expression of both Together, these data suggest that the level of stress influences the level of target derepression after anti-miR-208a treatment, and that target derepression for this subset of these genes is conserved in a large animal model of MI.In an effort to explore the mechanism behind the increased target derepression under stress conditions, we used neonatal rat ventricular myocytes (NRVMs) exposed to isoproterenol (ISO) or phenylephrine (PE),AntimiRs have shown to be efficacious in establishing therapeutic benefit under multiple disease conditions. Mouse genetics has shown us that miRNA functions are often more pronounced during disease and that the regulated mRNA targets can depend on the disease condition.Our data indicate that anti-miR treatment under diseased conditions results in derepression of a larger number of target genes than anti-miR treatment under baseline conditions. Additionally, we show that the strength of target regulation is increased during stress in rats, which we can confirm in a porcine model of ischemic injury.There are many potential explanations for our observations. The impact of stress on anti-miR efficacy might be because of a change in anti-miR activity or a direct effect of stress on target regulation. Stress might increase miR-208a inhibition by an increase in cellular uptake of anti-miR-208a, endosomal escape of anti-miR-208a, a change in cellular localization of either the anti-miR or miRNA that changes their interaction, or even an expressional change in long non-coding RNAs (lncRNAs) during stress that might influence the effect of the anti-miR by changing the cellular level of miR-208a.The effect of an miRNA on its target depends on the ratio of miRNA to targetAlthough the anti-miRs are highly specific in targeting an miRNA, we can currently not exclude that the difference in target regulation is also partly due to a change in abundance of additional miRNAs that can also regulate the measured transcripts.Although the level of target regulation is likely also under the influence of mRNA abundance and activity of the miRNA itself, our in vitro data show that the stronger effect of anti-miR treatment under diseased conditions could potentially also be caused by an enhanced uptake of the compounds when the cells become stressed. Several attempts to confirm this in vivo by injecting labeled anti-miR-208a failed, likely because of inefficient targeting of the heart.Our study also showed that the genes that are derepressed by an anti-miR partially depend on the disease driver. This effect is probably due to the fact that the target miRNA is regulating a different gene set when divergent signaling pathways and genes are activated in response to different stressors. Hypertension-induced cardiac remodeling will activate a different gene program than cardiac remodeling in response to MI. Therefore, it seems fair to assume that the divergence in gene regulation caused by the presence of stress or under different disease conditions is largely due to the availability of the mRNA targets present in the cells.It is necessary to measure target derepression to demonstrate efficacy of target engagement by anti-miRs in vivo. Although miRNA-induced changes in gene expression can occur at both the mRNA and the protein levels, the majority of changes occur because of mRNA destabilization.Although the exact mechanisms of enhanced target regulation under diseased conditions remain to be defined, our observations could have far-reaching implications for the clinical use of anti-miRs as novel therapeutics. The stronger pharmacological effects of anti-miRs during disease and the fact that disease etiology determines the therapeutic outcome of an anti-miR will be important for assessing the therapeutic dose and predicting the therapeutic effect in patients. Although this is important information to take along in developing an optimal therapy, both the importance of miRNAs and the potency of anti-miRs support enthusiasm for further pursuing these gene expression regulators as novel therapeutic candidates.Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at miRagen Therapeutics , the institutional review committee of the Royal Netherlands Academy of Arts and Sciences (KNAW) (anti-miR studies in angiotensin II-treated rats), or the Servier Research Institute ethical committee (porcine studies), and they comply with the federal and state guidelines concerning the use of animals and research as defined by the Guide for the Care and Use of Laboratory Animals  or with national animal welfare laws under a project license of the Dutch or French government.Adult Wistar (Charles River) male rats were anesthetized with isoflurane at 5% for 2–3 min, intubated, and ventilated using a rodent ventilator . Isoflurane was maintained at 1.5%–2.5%. Surgery was performed on a heated plate to maintain body temperature at 37°C; body temperature was monitored via rectal probe. The heart was reached through a left-sided thoracotomy between ribs 4 and 5; the left anterior coronary artery was ligated using a 7-0 silk suture. Successful ligation was confirmed by loss of color of the myocardium distal to the ligation. The chest wall and skin were closed, and rats were allowed to recover. Once awake, they were removed from ventilator and moved to a warm recovery cage. Once fully ambulatory, they were returned to normal housing and given 0.01 mg/kg buprenorphine subcutaneously. Sham-operated rats were subjected to the same procedure, with the exception that no ligation of the coronary artery was performed. After surgery the rats were kept for 8 weeks before sacrifice and tissue collection.Male Dahl rats (Harlan) were maintained on a 6% NaCl diet for 8 weeks starting at 7 weeks of age.Adult male Sprague-Dawley rats received angiotensin II  for 8 weeks by osmotic minipumps . Rats were anesthetized with 4%–5% isoflurane and maintained with 1%–2% isoflurane supplemented with oxygen. Surgery was performed on a heated plate to maintain body temperature at 37°C. A subcutaneous pocket on the back of the rats (interscapular) was created using blunt-end scissors, after which the AngII-filled osmotic minipump was placed in this pocket and the wound was closed with wound clips . After 4 weeks the pump was replaced with a new AngII-filled pump. Control animals received the same procedure with pumps filled with vehicle .Göttingen minipigs (Ellegaard) were subjected to ischemia-reperfusion (IR) by a closed-chest approach and clinical cardiac catheterization techniques.The carotid artery was exposed and access gained by the Seldinger technique. Under fluoroscopy guidance, a JR3.5 catheter was advanced over the wire to the level of the coronary sinus and placed at the coronary ostium without full engagement. Good placement was confirmed with a bolus of contrast agent (Telebrix) to visualize the left main, circumflex, and anterior descending coronary. Heparin (300 UI/kg i.v.) was given just before left anterior descending artery (LAD) occlusion to prevent clotting. A guide wire was advanced into the LAD followed by a balloon catheter (3.5 × 15–20) that subsequently was inflated at 4–6 atmosphere (atm) to ensure occlusion of the LAD for 150 min. After the 150 min occlusion, the balloon catheter was deflated and slowly removed. A bolus of lidocaine (2 mg/kg i.v.) and nitroglycerin (40 μg/kg intracoronary) was administered to avoid vasospasm and arrhythmia. During the recovery period, buprenorphine (50 μg/kg intramuscularly [i.m.]) was given for analgesia. After waking up under a warming lamp the pigs were extubated, and butorphanol  and amoxicillin  were given for post-procedural care.AntimiRs (designed and synthesized by miRagen) were dissolved in saline and delivered by subcutaneous injection. Used anti-miRs are LNA-DNA mixmers; these 16-mers are complementary to miR-208a specifically and have high nuclease resistance, as well as high duplex melting temperature.Total RNA was extracted from left ventricular (LV) tissue or cultured NRVMs with TRIzol according to the manufacturer’s protocol (Invitrogen).Gene expression profiling was performed by a service provider (MOgene) on Agilent SurePrint G3 Rat Gene Expression, 8 × 60 microarrays. Sample integrity was assessed with Agilent Bioanalyzer prior to microarray analysis. Data were analyzed using Array Studio software. Significantly regulated genes were defined using a Benjamini-Hochberg false discovery rate (BH-FDR) corrected p value cutoff of ≤0.05 to control for multiple testing. Differential gene expression reflects statistically significant expression in the treatment group compared with the saline-treated group. Hierarchical clustering was performed using the software program R using an agglomerative metric with Euclidean distance. For this study, genes containing a 6-, 7-, or 8-mer miR-208a binding site in their 3′ untranslated region were considered as potential miR-208a target. For these target genes, an average fold change in gene expression after anti-miR-208a treatment over control was calculated for each animal model (n = 4 per group). The genes that show a significant derepression after anti-miR-208a were used to generate the heatmaps shown in −ΔΔCT method and expressed as mean fold change ± SEM.cDNA was synthesized from 400–1,000 ng of total RNA extracted from tissue or cells using the iScript cDNA Synthesis Kit (Bio-Rad) for genes and miScript II RT Kit (QIAGEN) for miRNAs. Real-time PCR was performed to analyze the expression levels of individual mRNAs/miRNAs, using a specific set of primers  and iQ SNeonatal rat ventricular cardiomyocyte (NRVM) cultures were isolated by enzymatic dissociation of neonatal rat hearts, as described previously.For stress experiments, isoproterenol  or phenylephrine  was added to the (serum-free) culturing medium. For subsequent anti-miR treatment, the Cy3-labeled anti-miR-208a  was added to the medium after 8 hr of culture (passive delivery), without removing the stressor. Cells were then cultured for a further 16 hr before fixing and imaging.NRVMs cultured on coverslips were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 (Sigma-Aldrich), blocked with 1% fish gelatin (Sigma-Aldrich), and stained with an anti-α-actinin primary antibody  and an Alexa 488-conjugated secondary antibody . Coverslips were then mounted with ProLong Gold antifade reagent with DAPI . Cells were imaged using a Leica TCS SPE. Cross-sectional area (CSA) and uptake of Cy3-labeled anti-miR (as indicated by Cy3 fluorescence intensity) were measured using ImageJ. For all experiments where fluorescence intensity was quantified, laser and detector settings were kept consistent across the experiment. Quantifications were based on 17–43 cells per biological replicate, collected from 10 fields per replicate, for 8–12 replicates per condition.Values are presented as mean ± SEM. Outliers were identified and excluded using a Grubbs’ test . Statistical significance was evaluated using an unpaired t test for comparisons between two groups, using GraphPad Prism software. The kernel density plot was generated using R, as were the regression line and matching confidence interval in Conceptualization, J.E.C.E., C.J.D., and E.v.R.; Formal Analysis, J.M.L., J.E.C.E., and C.J.D.; Investigation, J.E.C.E., C.J.D., A.G.S., R.L.M., H.M.S., A.L.J., M.I., and S.C.; Resources, A.G.S., R.L.M., H.M.S., A.L.J., M.I., and S.C.; Writing – Original Draft, J.E.C.E., C.J.D., and E.v.R.; Writing – Review & Editing, J.E.C.E., C.J.D., and E.v.R.; Visualization, J.E.C.E. and C.J.D.; Supervision, E.v.R.; Funding Acquisition, E.v.R.J.M.L., A.G.S., R.L.M., H.M.S., and A.L.J. are employees of miRagen Therapeutics. M.I. and S.C. are employees of Servier. E.v.R. is a scientific co-founder and member of the Scientific Advisory Board of miRagen Therapeutics, Inc.


The uptake inhibition experiments also demonstrate that our nanoparticles were incorporated via the classic clathrin-mediated endocytosis pathway. These results indicate that hemoglobin nanoparticles formed by self-assembly are a promising drug delivery system for cancer therapy.Hemoglobin is a promising drug carrier but lacks extensive investigation. The chemical conjugation of hemoglobin and drugs is costly and complex, so we have developed curcumin-loaded hemoglobin nanoparticles (CCM-Hb-NPs) via self-assembly for the first time. Using the acid-denaturing method, we avoid introducing denaturants and organic solvents. The nanoparticles are stable with uniform size. We have conducted a series of experiments to examine the interaction of hemoglobin and CCM, including hydrophobic characterization, SDS-PAGE. These experiments substantiate that this self-assembly process is mainly driven by hydrophobic forces. Our nanoparticles achieve much higher cell uptake efficiency and cytotoxicity than free CCM solution  Abraxane® was approved by US Food and Drug Administration (FDA) in 2005 for its dramatically enhanced water-solubility and maximum tolerated dose [Being a major component of organisms, proteins have been the preferred biomaterials for drug delivery vehicles . Firstlyted dose .in vivo. Hemoglobin has also been used widely as a blood substitute in clinical or preclinical research [et al. had developed hemoglobin-ribavirin conjugates for targeting therapy purpose, but only six to eight ribavirin moieties attached per hemoglobin (less than 3% drug loading) [Hemoglobin (Hb) is roughly spherical, with a diameter of nearly 5.5 nm and a molecular weight of 64.5 kDa . It is aresearch ,10,11,12research ,14. Comploading) .® is prepared by emulsification through high-pressure homogenization followed by rotary evaporation, and the active agent paclitaxel (PTX) is dissolved in methylene chloride. This method is using homogenization to break up the intermolecular disulfide bonds between the sulfhydryl groups and make the resulting solution stable, with a particle size of 50–220 nm [et al. used β-mercaptoethanol (β-ME) and dithiothreitol (DTT) as denaturing reagents to successfully encapsulate paclitaxel and curcumin (CCM) into human serum albumin and demonstrated that the hydrophobic interaction was the main force of the assembly process of albumin nanoparticles [To obtain protein-based drug-loaded nanoparticles, self-assembly of unfolded or partially unfolded proteins is convenient and cost-effective compared to chemical synthesis. Taking albumin as an example, Abraxane0–220 nm . Anotherarticles ,16,17. Nin vitro. It was reported that hemoglobin exhibited multistep unfolding transitions as pH declined [et al. demonstrated that the CD spectra of Hb at pH 1.9 showed a large departure from those at pH > 3 [Hemoglobin undergoes structural changes at different pH values declined ,19. Betwt pH > 3 . In thist pH > 3 ,22. AddiThe exposure of the hydrophobic area inside the hemoglobin was demonstrated by the Tryptophan (Trp) fluorescence and 1-(anilinon) aphthalene-8-sulfonic acid (ANS) ,24. Trp Then ANS was used to observe the change in the exposed hydrophobic region of hemoglobin. ANS is minimally fluorescent in polar environments, such as aqueous solutions, but its fluorescence dramatically increased in nonpolar environments, including binding with the hydrophobic area of the protein . ResultsOnce the tryptophan residue in the denatured hemoglobin is exposed to the solvent, the tryptophan fluorescence is quenched in the presence of ANS. The possible mechanism is the fluorescence resonance energy transfer (FRET) between the tryptophan residue and the ANS molecules due to overlap of the tryptophan emission and ANS excitation spectrum. As shown in We prepared CCM-Hb-NPs using the acid-denaturing method. As shown in The major properties of CCM-Hb-NPs were summarized in et al. demonstrated that the CD (circular dichroism) spectra of Hb at pH 1.9 showed a large departure from those at pH > 3 [According to t pH > 3 . In addiTo investigate the driving forces of the formation of nanoparticles, we used ethyl acetate to extract CCM from CCM-Hb-NPs based on the theory of similarity and intermiscibility. After adding CCM-Hb-NPs aqueous solution to ethyl acetate, there would be a demarcation line between the two layers . As showIn order to confirm that non-covalent interactions stabilized our nanoparticles, we performed the SDS-PAGE of CCM-Hb-NPs and native hemoglobin. SDS could break hemoglobin into dimer and monomer form, but could not break up covalent bonds of proteins. So SDS-PAGE could be performed to substantiate the non-covalent cross-linking of hemoglobin during the nanoparticle formation process . As showTo further identify the type of non-covalent interaction between proteins and CCM, we used ethanol to dissolve the nanoparticles. Ethanol could weaken the hydrophobic interaction . When divs. 5.98 ± 0.98), indicating that better cell inhibition could be achieved in the CCM-Hb-NPs group than in the free CCM group  after incubation for 8 h. In the CCM-Hb-NPs group, a larger amount of CCM was accumulated into the cytoplasm of the MCF-7 cells compared to the free CCM A. ResultCM group B.vs. 2.38 ± 0.29). The results indicated that temperature played an important role in the cell uptake of CCM-Hb-NPs  and indomethacin (a caveolae-mediated endocytosis inhibitor) on cellular uptake efficiency . The celM-Hb-NPs .The cellular uptake efficiency of CCM at 37 °C was significantly inhibited by sucrose (a clathrin-mediated endocytosis inhibitor). In contrast, indomethacin, a caveolae-mediated endocytosis inhibitor, failed to inhibit the uptake of CCM. These results strongly indicated that our nanoparticles were internalized into the cytoplasm via the clathrin-mediated endocytosis pathway.The cytotoxicity of free CCM and CCM-Hb-NPs was also investigated in MCF-7 cells A. After in vivo, so further animal study should be conducted to evaluate the safety and side effects of CCM-Hb-NPs [Although CCM-Hb-NPs showed enhanced cytotoxicity compared with free CCM, hemoglobin had potential toxicity because of the heme part. Therefore, the cytotoxicity of hemoglobin alone was also conducted. Hemoglobin showed no toxicity on the MCF-7 cell line even at the concentration of 9 mg/mL at different pH values B. HoweveM-Hb-NPs .Human Hemoglobin (99%) was purchased from Sigma-Aldrich. Curcumin (CCM) (purity ≥ 99%) was purchased from Zelang Medical Technology Co., Ltd.  and cell counting kit-8 (CCK-8) was purchased from Dojindo . The human breast cancer cell line (MCF-7) was purchased from Shanghai Institute of Cell Biology . Ethanol, ethyl acetate, 8-anilino-1-naphthalenesulfonic acid (ANS), salts, buffers and acids were analytic reagents and purchased from WanQing Chemical Classware Instrument Co., Ltd. .First, 80 mg of hemoglobin was dissolved in 20 mL water at room temperature. Then the pH of the protein solution was adjusted to 4.0 using 2 M HCl. Ten minutes later, CCM  was slowly added to protein solution to prepare the nanoparticles. After that, the nanoparticle solution was ultrafiltered three times to remove free CCM and ethanol, and about 3 mL of final concentrated solution was obtained for further use.Spectrophotometric method was used for the analysis of CCM amount in NPs. Briefly, the CCM-Hb-NPs were decomposed by acetate. After that, the mixed solution was diluted in 50-fold volume of chloroform and sonicated for 15 min to extract the CCM completely. The drug concentration was measured by reading the absorbance at λ = 419 nm using UV spectrophotometer  and calculating from the standard curve of CCM in chloroform. The concentration of hemoglobin was detected by Coomassie brilliant blue.The drug loading percentage (DL) and encapsulation efficiency (EE) were calculated according to:Acid unfolding of human hemoglobin was performed by incubating the protein at pH 2.5–6.6 for 0.5 h . The structure change of hemoglobin was detected by UV-visible spectroscopy. The second-structure of hemoglobin before and after forming nanoparticles was determined by circular dichroism.The average particle size and size distribution were determined by dynamic light scattering . The data were obtained from the average of triplicate measurements. The solution of CCM-Hb-NPs was placed on a 200-mesh copper grid coated with carbon. After deposition, the grid was air-dried and the morphology of nanoparticles was observed by transmission electron microscopy .The exposure of tryptophan residues was detected by monitoring the fluorescence intensity of tryptophan in hemoglobin using an RF-5301PC fluorescence spectrophotometer  at 0.5 nm intervals. Tryptophan emits fluorescence at 300 to 400 nm, under excitation at 280 nm. The hydrophobic dye ANS was used as an indicator of exposure of hydrophobic clusters for its high affinity to hemoglobin. Then 40 μM ANS was added to hemoglobin at pH 3.3, 4.4, 5.4 and 6.6, respectively. The fluorescence change of ANS (40 μM) alone was also conducted at pH 4.1, 5.3, 6.1 and 7.4. The fluorescence at 400 to 550 nm was recorded after 15 min  at 0.5 nm intervals, with 380 nm as the excitation wavelength.Hemoglobin solutions (0.2 mg/mL) at pH value of 4.1 were incubated with different amounts of CCM , and the fluorescence of the nanoparticles and free hemoglobin of the same concentration were determined using a fluorescence microplate  The excitation wavelength was 280 nm, and the emission spectrum ranged from 310 nm to 400 nm.Fluorescence resonance energy transfer studies were carried out using fluorescence spectrophotometer F-7000 . Hemoglobin solution was adjusted to 4.2 by 2 M HCl. Protein samples containing different amounts of ANS (40 μM) were equilibrated for 20 min at room temperature before recording. The excitation wavelength was 280 nm, and the emission was recorded from 300 to 550 nm. Hemoglobin concentration was 0.18 mg/mL for experiments.During the nanoparticle preparation process, the stability time of solution is a major parameter to evaluate whether nanoparticles were well prepared. “Stability” here means that during the process of nanoparticles preparation, if hemoglobin could load CCM well, the solution would show homogeneous state and no aggregation could be observed. If nanoparticle size was larger than 1 μm, we think that nanoparticles had shown precipitation and were not stable. CCM-Hb-NPs were prepared over a pH range from 4 to 7. At four different pH values , the particle size of nanoparticles was measured by dynamic light scattering . The precipitate time of nanoparticles was also recorded. The size change of CCM-Hb-NPs diluted in PBS (pH 7.4) in 48 h was also detected by DLS.The 0.2 mL CCM-Hb-NPs solution was mixed with 3 mL ethyl acetate through lightly shaking, and the concentration of CCM in ethyl acetate was measured by UV spectrophotometer  and calculated from the standard curve of CCM in ethyl acetate.The particle size of nanoparticles added with four concentrations of ethanol  was measured by dynamic light scattering .Hemoglobin of CCM-Hb-NPs was separated on 12% SDS-PAGE gel. Approximately 10 μL of CCM-Hb-NPs and native hemoglobin were loaded per well. The gel was stained with Brilliant Blue R-250 staining solution  at 37 °C for 1 h in a shaker at 100 rpm and later destained by destaining solution (Bio-Rad) for up to 3 h at 37 °C.4 cells/well) and cultured overnight. Then the cells were exposed to 10 μg/mL of free curcumin or CCM-Hb-NPs (same dose of curcumin). After culturing for 8 h, the cells were washed twice with PBS and fixed with fresh 4% paraformaldehyde for 10 min. The nuclei of cells were then stained with DAPI for 15 min. Finally, the cells were imaged by confocal laser scanning microscopy  in FITC (for CCM) and DAPI channel, respectively.The cellular uptake of CCM-Hb-NPs could be determined by detecting the fluorescence of CCM using Olympus FV1000 confocal laser scanning microscope . Briefly, the MCF-7 cells were seeded in 12-well plates  or 100 μM indomethacin (inhibitor of caveolae-mediated uptake) or at 4 °C. Then the cells were exposed to 10 μg/mL free curcumin or CCM-Hb-NPs (same dose of curcumin). After further culturing for 8 h, the cells were washed twice with PBS and fixed with fresh 4% paraformaldehyde for 10 min. The nuclei of cells were then stained with DAPI for 15 min. The cells were imaged by confocal laser scanning microscopy in FITC channel (for CCM) or DAPI channel, respectively.3 cells per well in 100 μL media) and cultured overnight. The culture medium was replaced with a fresh one containing equivalent doses of free CCM and CCM-Hb-NPs of 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 60 μM, respectively. After incubation for 24 h, the culture medium was removed and cells were washed with PBS. Then 10% (v/v) CCK-8 solution in fresh medium was added and cells were incubated at 37 °C for 2 h. The absorbance of the solutions was then measured at 450 nm by a microplate reader . Cells cultured in PBS were the control group. Inhibition Rate =  × 100%. Multiple groups were compared using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for post hoc comparisons. Statistical significance was set at p < 0.05.The cytotoxicity of CCM-Hb-NPs against human breast cancer cell line MCF-7 was measured by Counting Kit-8 (CCK-8) assay. CCK-8 was used to determine the living cells. Briefly, the cells were seeded in 96-well plates (5 × 10in vitro and in vivo, it has a significant clinical prospect as an anti-cancer compound. Our CCM-Hb-NPs enhanced CCM solubility and achieved higher anti-tumor activity compared with free CCM. Hemoglobin may be a good carrier for CCM, as hemoglobin could target to CD163-overexpressed macrophages and monocytes. In further study, the anti-tumor efficacy will be examined in vivo.In this study, we firstly proposed hemoglobin as a drug carrier to encapsulate the lipophilic drug CCM via the self-assembly method. In addition, through extensive investigation of the formation mechanism of CCM-Hb-NPs, we concluded that hemoglobin assembled into nanoparticles driven by hydrophobic interactions. Firstly, we found that the fluorescence intensity of Trp in hemoglobin would augment in acidic environment, which indicated that low pH would expose the hydrophobic domain inside the protein. To further examine its hydrophobic pocket, we used the hydrophobic probe ANS to mark the hydrophobic region and measured the fluorescence of ANS. Results showed that the hydrophobic region was exposed as we speculated, in accordance with the Trp fluorescence augment. Then we successfully incorporated the lipophilic CCM into hemoglobin in pH 4 to 5.5, and the binding region was suggested to be the hydrophobic area of hemoglobin. The fluorescence of Trp was quenched as soon as CCM was added. The following extraction experiment using ethyl acetate demonstrated that CCM-Hb-NPs were formed through non-covalent bonds. Ethanol, which could weaken hydrophobic interactions, was also used to interact with the nanoparticles. Results indicated that the incorporation of CCM into nanoparticles should be driven by hydrophobic forces. After demonstration of the formation mechanism, we also performed cell cytotoxicity and uptake experiments, and the results confirmed that both the uptake efficiency and cytotoxicity were significantly enhanced. The endocytosis-inhibiting experiment proved that CCM-Hb-NPs were internalized through a clathrin-mediated endocytosis. Because CCM inhibits proliferation of various tumor cells both


Determination of the impact of visceral obesity and epicardial adipose tissue thickness on stimulating growth factor levels during hospitalization for myocardial infarction is of potential importance for predicting outcomes and assessing the development of cardiofibrotic changes associated with maladaptive myocardial remodeling. In this study, we aimed to investigate the relationships between epicardial adipose tissue thickness, adipokine profiles, and the stimulating growth factor 2/interleukin-33 signaling system during hospitalization for myocardial infarction, and with the cardiac fibrosis extent 1-year post-MI in patients with visceral obesity.Eighty-eight patients with myocardial infarction were grouped based on their visceral obesity. Serum leptin, adiponectin, stimulating growth factor 2, and interleukin-33 levels were measured on days 1 and 12 and at 1 year. The epicardial adipose tissue widths and the cardiac fibrosis areas were measured on day 12 and at 1 year.Visceral obesity was associated with epicardial adipose tissue thickness increases, adipokine imbalances, elevated leptin levels, and lower adiponectin levels during early hospitalization, and cardiac fibrosis development. Patients without visceral obesity had higher interleukin-33 and stimulating growth factor 2 levels during early hospitalization and lower cardiac fibrosis rates. Epicardial adipose tissue thickness was positively associated with cardiac fibrosis prevalence and interleukin-33 levels and negatively associated with stimulating growth factor 2 levels. The cardiac fibrosis extent was negatively associated with interleukin-33 levels and positively associated with stimulating growth factor 2 levels.Increases in epicardial adipose tissue thickness are associated with cardiac fibrosis development 1-year post-myocardial infarction and are higher in patients with visceral obesity. The metabolic activity of the epicardial adipose tissue is associated with elevated interleukin-33 and reduced stimulating growth factor 2 levels. Adipose tissue, particularly its visceral component, is central to the initiation of many pathophysiological processes, including cardiac fibrosis development . The infStimulating growth factor 2 (ST2) is the most promising marker of early myocardial remodeling. It is expressed on the cardiomyocytes in response to biomechanical stress . ST2 is This study aimed to assess the relationships between EAT thickness, the adipokine profiles, and the ST2/IL-33 signaling system during hospitalization for MI, and the degree of cardiac fibrosis at 1-year post-MI in patients with visceral obesity.The study was performed at the Federal State Budgetary Institution’s Research Institute for Complex Issues of Cardiovascular Diseases. The study’s protocol was approved by the local ethics committee. Patients were included in the study after they provided written informed consent.Eighty-eight patients with MIs, comprising 65 men and 23 women, were included in the study. The patients’ mean age was 58 (range: 52.0–63.5) years. An MI diagnosis was verified using the diagnostic criteria from the Russian Society of Cardiology (2007) and those from the European Society of Cardiology, American College of Cardiology Foundation, American Heart Association, and the World Heart Federation Task Force for the Redefinition of Myocardial Infarction . The con2, and a VAT to SAT ratio ≥ 0.4 [n = 29) (mean age: 56.0 [range: 51.5–63.5] years), and Group 2 comprised patients with visceral obesity (n = 59) (mean age: 58.50 [range: 53.0–63.0] years). Table The VAT and subcutaneous adipose tissue (SAT) quantities were measured using multislice computed tomography (CT) imaging and a 64-row multidetector CT system  to confirm the presence of visceral obesity, which was defined as VAT > 130 cmThe EAT thickness was measured using magnetic resonance imaging (MRI)  with a magnetic field strength of 1.5 T. A T1-weighted rapid pulse sequence combined with ECG synchronization was used . The sections were orientated to the left ventricular short axis. Using standard instruments, a layer of adipose tissue was measured at the levels of the outer edge of the myocardium to the visceral pericardial sheet along the anterior wall of the right ventricle (RV) and along the posterior wall of the left ventricle (LV) . Imaging of the cardiac fibrosis that was assessed using delayed contrast enhancement, was performed 6 min after the contrast medium was introduced using a T1-weighted pulse sequence . The sections were orientated to the left ventricular short axis. The DICOM images were processed and analyzed using Segment software (Medviso). The percentage of cardiac fibrosis within the myocardium’s total mass was automatically calculated when myocardial scarring was present.All patients underwent contrast-enhanced MRI (ExcelartAtlas 1.5 MRI imager) with a magnetic field strength of 1.5 T at 1-year post-MI to assess myocardial scarring. Paramagnetic gadolinium medium was used (0.5 mmol/mL), and the contrast medium (0.2 mL/kg) was injected automatically into the cubital vein at 3 mL/s using a syringe injector (OptistarThe serum levels of leptin and its soluble receptor (sOB-R), adiponectin, IL-33, and ST2 were measured on days 1 and 12, and at 1-year post-MI, using commercially available enzyme-linked immunosorbent assay kits . Leptin sensitivity was evaluated using the free leptin index (FLI), which was defined as the ratio of the total leptin concentration in ng/mL to the sOB-R concentration in ng/mL. Leptin resistance was defined as an FLI > 0.25. The leptin to adiponectin ratio determined the presence of an adipokine imbalance.p < 0.05 was considered statistically significant.The statistical analyses were performed using Statistica software, version 6.1  and SPSS, version 17.0 for Windows . The Kolmogorov–Smirnov test was used to assess the data distributions. The data are presented as the medians and the 25th and 75th quartiles. The Mann–Whitney U test was used to compare the independent groups with skewed distributions. A Fisher’s exact test with a two-sided confidence probability was used to analyze the frequencies of the categories in two independent groups. A value of p > 0.05). Patients with visceral obesity tended to be heavier, and to have hypercholesterolemia, angina pectoris, and type 2 diabetes mellitus. Chronic heart failure was more regularly recorded in patients without visceral obesity. The study groups did not differ significantly regarding the hospitalization duration and the therapies administered , including arterial hypertension, smoking, and a family history of CAD . During follow-up, the leptin levels declined in both study groups, but they did not match the reference values. On day 12 post-MI, the leptin levels in patients with visceral obesity were 3.83-times higher compared to the control individuals and 2.55-times higher compared to patients without visceral obesity. There was no statistically significant difference between the study groups regarding the leptin levels (p > 0.05). At 1-year post-MI, the leptin levels were 1.5-times higher in patients with visceral obesity compared to patients without visceral obesity. The sOB-R levels were similar in both study groups, but at 1-year post-MI, they increased by 10% in the patients with visceral obesity compared to the control individuals and patients without visceral obesity (p < 0.05). The FLIs > 0.25 suggested the presence of leptin resistance in both study groups during the follow-up period. During hospitalization, the adiponectin levels declined by 35.7% in patients with visceral obesity and by 14.6% in patients without visceral obesity compared to the control individuals (p < 0.05). In patients without visceral obesity, the adiponectin levels increased rapidly and reached levels that were similar to those present in the control individuals on day 12 post-MI. In patients with visceral obesity, the normalization of the adiponectin levels was observed at 1-year post-MI. During the follow-up period, the leptin to adiponectin ratios in patients with visceral obesity exceeded those calculated for the control individuals and patients without visceral obesity.Compared with the control individuals, the acute MI phase was accompanied by elevated leptin levels in both patient groups Fig. . The lepp = 0.03) at 1-year post-MI. The sizes of the areas of cardiac fibrosis did not differ between patients with and without visceral obesity (10.1% vs. 12.9%). Cardiac fibrosis was absent in more of the patients without visceral obesity than those with visceral obesity . Cardiac fibrosis affected 1–5% of the myocardium in 33.3 and 22.7% of the patients with and without visceral obesity, respectively. More severe cardiac fibrosis affecting 20% of the myocardium was more common in patients without visceral obesity compared with those with visceral obesity (p = 0.03). The prevalence of cardiac fibrosis was higher in patients with visceral obesity compared to patients without visceral obesity at 1-year post-MI, and this difference was statistically significant.Cardiac fibrosis was diagnosed in 79.1% of patients with visceral obesity and in 72.9% of patients without visceral obesity (p < 0.05). On day 12 post-MI, the ST2 levels were 3-times lower in patients without visceral obesity and 1.6-times lower in patients with visceral obesity compared with those on day 1 post-MI (p < 0.05). At 1-year post-MI, the ST2 levels in the study groups did not match those in the control individuals.Differences were found in relation to the ST2/IL-33 signaling system depending on the presence of visceral obesity. On day 1 post-MI, the ST2 levels were 3.5-times higher in patients without visceral obesity compared to controls, and they were 2.4-times higher in patients without visceral obesity compared to patients with visceral obesity (p < 0.05). On day 12 post-MI, a slight but statistically significant decrease in the IL-33 levels occurred in both study groups, but these levels did not match the control group’s levels. The IL-33 levels had increased by twofold in the study groups at 1-year post-MI compared with the IL-33 levels on day 1 post-MI and by 17-fold compared with the IL-33 levels in the control individuals (p < 0.05)  and RV  levels. There was no correlation between the EAT thickness and the adiponectin levels. Negative correlations between the leptin levels at 1-year post-MI and the EAT thickness at the LV and RV levels  were determined in the patients with visceral obesity, but not in those without visceral obesity. Negative correlations were determined between the EAT thickness and the FLIs during hospitalization  and at 1-year post-MI . Positive correlations between the IL-33 levels, the ST2/IL-33 signaling system, and the EAT thickness were determined in patients with and without visceral obesity during hospitalization  and at 1-year post-MI . The ST2 levels were negatively correlated with the EAT thickness .The correlation analysis Figs. , 5 deterThus, visceral obesity in patients with MI was associated with an increased EAT thickness, an adipokine imbalance, elevated leptin levels, reduced adiponectin levels during early hospitalization, and with the development of cardiac fibrosis. Higher IL-33 and ST2 levels in patients without visceral obesity during early hospitalization were followed by a lower prevalence of cardiac fibrosis after discharge. The EAT thickness was positively correlated with the degree of visceral obesity and the IL-33 levels, and negatively correlated with the ST2 levels.EAT is a unique and multifaceted fat depot that exerts local and systemic effects –16, and This study’s results showed a direct correlation between EAT thickness at the RV and LV levels and the degree of cardiac fibrosis. However, a key role for leptin in cardiac fibrosis was not confirmed, and there was no correlation between the leptin level and the degree of cardiac fibrosis, which may have been a consequence of the presence of leptin resistance that was confirmed by the high FLI. This notion is further supported by the negative correlation between the sOB-R level and the degree of cardiac fibrosis.The adiponectin levels in MI patients with visceral obesity were fairly low during hospitalization, and they were similar to those in patients without visceral obesity. The leptin to adiponectin ratio can indicate an adipokine imbalance , and it ST2 is one of the most promising biomarkers of cardiac fibrosis . Weir etThe correlation analysis determined the presence of an inverse relationship between the ST2 levels and EAT thickness at the RV and LV levels. There are no published data that describe the synthesis of ST2 in adipose tissue. The endothelial cells are the main source of ST2, and it is possible that the increase in the ST2 levels associated with MI is not related to EAT and that it may be a consequence of endothelial activation, which is common in MI.ST2 mainly potentiates the effects of IL-33. During hospitalization, the IL-33 levels increased by eightfold and 9.2-fold in patients with and without visceral obesity, respectively. At 1-year post-MI, the IL-33 levels were 17-times higher in all of the patients compared with controls. IL-33 has antihypertrophic and antifibrotic effects, and an increase in the IL-33 level may protect against the development of cardiac fibrosis. Hence, the elevated IL-33 levels in MI patients without visceral obesity may have limited cardiac fibrosis compared with that in MI patients with visceral obesity. Of the MI patients without visceral obesity, 27% did not have cardiac fibrosis, and 21% of MI patients with visceral obesity did not have cardiac fibrosis. The degree of fibrosis was inversely related to the IL-33 levels and the ST2/IL-33 signaling system, which confirms the antifibrotic effects of IL-33 . The IL-This study’s findings suggest that EAT may produce favorable levels of protective IL-33. In addition to its adverse effects, EAT serves as a local energy supplier, a buffer against excess free fatty acid levels, and as a source of protective cytokines and adipokines, including IL-10. Our data suggest that compared with patients without visceral obesity, an increase in the EAT thickness in patients with visceral obesity is accompanied by lower levels of endothelial IL-33 and ST2 during hospitalization and a smaller area of cardiac fibrosis (1–5% tissue damage). An increase in the levels of protective IL-33 cannot entirely prevent the development of cardiac fibrosis in these patients. Epicardial adipocytes can synthesize their own adipo-fibrokines, including activin A and MMP8, and Venteclef et al. [In the previous study, we showed that EAT-derived and circulating secreted frizzled-related protein 4 (SFRP4) expression levels are increased in patients with CAD and in epicardial adipocytes of CAD patients, the concentrations of leptin, tumor necrosis factor-α, and IL-1, were higher, while the levels of adiponectin, IL-10, and FGF-β were lower than those in subcutaneous adipocytes , 24. BouAn increase in EAT thickness was closely related to the development of cardiac fibrosis at 1-year post-MI. The EAT was thicker in patients with visceral obesity who had more pronounced adipokine imbalances, that is, lower levels of protective adiponectin, higher leptin levels, and leptin resistance. The metabolic activity of EAT was associated with an increase in the IL-33 levels and a decrease in the ST2 levels. The degree of cardiac fibrosis was negatively related to the IL-33 levels and positively related to the ST2 levels.


Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pig pathogen, belonging to the class Mollicutes. It causes polyserositis, arthritis and cancers in vitro, increasing attention of the researchers. Currently, there is no available genetic tool to manipulate its genome. This study describes a development of oriC-plasmids harboring either large (pGEMT-LoriC) or minimum (pGEMT-MoriC) origin of replication (oriC) of M. hyorhinis along with tetracycline resistance marker.These plasmids were successfully transformed into M. hyorhinis with average transformation frequency of 1.5 × 10−4 and 2.0 × 10−5 transformants/CFU for pGEMT-LoriC and pGEMT-MoriC respectively, and were integrated at the chromosomal oriC as well as remained freely replicating. We also constructed a Mini-oriC-HT1 targeting plasmid by inclusion of hlyC arms and was used to inactivate hlyC at average frequency of 50%. The efficiency of hlyC inactivation was further improved (by 90%) when Mini-oriC-HT2 that contains E. coli recA was used. In both cases, hemolysin mutant bacteria diminished the ability to lyse mouse RBCs compared to wild-type (P < 0.001). OriC-plasmids described in this study may, therefore open the way for functional genomics in M. hyorhinis. Furthermore, this is a first study demonstrated the gene associated with a hemolytic phenotype in mycoplasmas. Mycoplasma hyorhinis (M. hyorhinis), is an opportunistic pathogen of pigs associating with respiratory tract infections and arthritis1. M. hyorhinis is most often detected as contaminants in cell cultures2. In addition, chronic M. hyorhinis infection has been found to play a role in the development of several types of cancers in vitro3, increasing attention of the researchers.M. hyorhinis has been completed and published several years ago4. However, studies to manipulate the genome of M. hyorhinis and understand the molecular pathogenesis suffers from the lack of genetic tools available for the pathogen.The genome sequence of M. hyorhinis, the transposon mutagenesis and its derivatives have been developed for genetic studies6. However, random integration of the transposon into the chromosome did not allow the specific gene targeting analysis.For many mycoplasmas including Mycoplasma mycoides subsp. capri7, Mycoplasma gallisepticum8, Acholeplasma laidlawii9 and Mycoplasma genitalium11. Indeed, in our laboratory, we have developed suicide plasmids and have been successfully used to inactivate hlyC in M. hyorhinis12.Suicide plasmids have also been used in Mollicutes to inactivate targeted genes via homologous recombination between the gene cloned in the plasmid and its homologues sequence in the host genome. Because of low recombination frequency that may cause by the low transformation efficiency, these vectors were applied only in very few mycoplasmas including Spiroplasma citri13, Mycoplasma pulmonis14, Mycoplasma mycoides subsp. capri, M. mycoides subsp. Mycoides small-colony type15, Mycoplasma capricolum subsp. capricolum16, Mycoplasma agalactiae17 and Mycoplasma gallisepticum18. The typical oriC-plasmid contains an origin of replication (oriC) along with tetracycline resistance marker and sufficient sequence homologous to chromosomal DNA6 to insert into the chromosome of the organism via homologous recombination. These oriC-plasmids have been used in some studies to disrupt targeted genes, complement mutants19 and also to express foreign genes20.Recently, replicative plasmids have been established for a number of Mollicutes including Mycoplasma mycoides subsp. capri GM12 (Mmc) genome using CRISPR/Cas9 system, has also been established and was used to disrupt mycoplasma glycerol-3-phosphate oxidase-encoding gene (glpO)21. However, cloning and assembly of a synthetic bacterial genome in yeast followed by a back transplantation into recipient bacterial cells is a challenge.More recently, in-Yeast engineering of oriC-plasmids have not been studied in M. hyorhinis. The aim of this study was to construct and evaluate the usefulness of these plasmids as genetic tools in M. hyorhinis. Here, the oriC-region of M. hyorhinis was characterized and used for the development of oriC-plasmids that replicates in M. hyorhinis.At present, the oriC-plasmids harboring large oriC-fragments tend to integrate into the oriC-region of the genomic DNA by homologous recombination between the gene fragment cloned on the plasmid and the corresponding site on the chromosome due to the sequence homology. The stability of the oriC-plasmids can be improved by utilizing a minimum oriC that reduces the possibility of the integration of the plasmid into the host chromosome22. Herein, a mini-oriC plasmid that could replicate and maintained stably extra-chromosomally has been developed and evaluated. This study also provides evidence of homologous recombination occurring in M. hyorhinis.In many cases, the 23. Since the HUB-1 genome sequence has a hlyC (NCBI Reference Sequence: WP_014582557.1) related sequence identified by automated prediction, it is hypothesized that this sequence is involved in hemolysis. Therefore, hlyC was chosen as the target gene for these experiments. We initially confirmed that, the supernatant of wild-type M. hyorhinis can lyse mouse RBCs  of homology with chromosomal DNA of hlyC flanking tetM used to transform M. hyorhinis, the tetM inserted into the chromosome and disrupted the hlyC. The supernatant of the hemolysin mutant strain diminished the ability to lyse mouse RBCs. Therefore, this manuscript demonstrates that hlyC may be involved in mouse RBC lysis.The hemolytic activity has been described in other mycoplasmasRecA plays an important role in DNA recombination and repair24. In M. mycoides subsp. capri, the efficiency of homologous recombination using suicide plasmids was enhanced by the inclusion of a recA (from E. coli) into the constructs25. Therefore, in this study, we further augmented the occurrence of the homologous recombination at the hlyC site by introducing recA into our targeting oriC-plasmids.M. hyorhinis has been long described. However, the transformation procedure and the oriC-plasmid systems developed in this study, have the potential to be used in targeted gene disruption, and may also in gene complementation and expression studies in this organism and possibly other mycoplasmas.The ability to transform oriC had previously been isolated from M. hyorhinis. The large oriC region (LoriC) of M. hyorhinis (1935-bp) which consisted of the complete dnaA and intergenic regions upstream and downstream of the dnaA (NCBI Reference Sequence: WP_020104468.1) were identified  of about 876-bp, only contains the regions upstream and downstream the dnaA , spiralin gene promoter 313-bp, large oriC (LoriC) 1935-bp, mini-oriC (MoriC) 876-bp, recA 1062-bp, hemolysin left arm (LA) 437-bp and hemolysin right arm (RA) 405-bp were prepared  was detected by tetM specific PCR  compared to 2.0 × 10−5 transformants per CFU for pGEMT-MoriC plasmid.Following transformation with oriC-plasmids containing LoriC fragment, have been shown to integrate into the host chromosome via homologous recombination due to the sequence homology with oriC-region. Here, we evaluated whether our pGEMT-LoriC had integrated into the M. hyorhinis genome, and also investigated the site at which the integration had occurred. Briefly, M. hyorhinis DNA extracted from transformed (at 5th passages) and untransformed control cultures, were subjected to the PCR analysis. Based on M. hyorhinis genomic sequence and pGEMT-LoriC plasmid, we designed integration primers . We also predicted the size of the PCR product when pGEMT-MoriC integrated at the oriC-site (2122-bp), where we get no PCR product  and was named pGEMT-MoriC. Since we aimed to evaluate whether pGEMT-MoriC plasmid could be used for hlyC inactivation. We cloned hemolysin left and right arms into the pGEMT-MoriC plasmid to flank tetM and the spiralin gene promoter and to yield Mini-oriC-HT1 targeting plasmid. The resultant mutant strain expected to confer resistance to tetracycline when added to the medium at the appropriate concentration. The following transformation with Mini-oriC-HT1 plasmid and plating in KM2-agar containing 2.5 µg/ml tetracycline, we observed the growth of tetracycline resistance colonies. Selected colonies sub-cultured in KM2 medium containing 5.0 µg/ml tetracycline and subjected to PCR analysis using hlyC flanking primers  and could be PCR amplified with hlyC flanking primers  controlled by the spiralin gene promoter into Mini-oriC-HT1 to yield Mini-oriC-HT2 targeting plasmid. The previous study indicated the usefulness of recA to enhance homologous recombination in M. mycoides subsp. capri7. Similar results were observed with our suicide vectors12. Following transformation with Mini-oriC-HT2 plasmid and sub-culture of 5 resistance colonies, we observed the predicted size of PCR product (3072-bp) in all the selected colonies indicating the insertion of tetM at the hemolysin site . No significant change in the phenotype of the wild-type and mutant colonies was observed  using single-cross primers  lacks the ability to lyse mouse RBS compared to wild-type bacterial supernatants 5, the usage of the host-vector systems for genetic studies of M. hyorhinis has not so far been implemented. Therefore, a host-vector system which can be utilized for M. hyorhinis genetic studies is a prerequisite. The electro-transformation conditions optimized in this study are suitable for M. hyorhinis. However, some factors such as the use of minimum inhibitory concentration of tetracycline, high voltage and the excess amount of plasmid DNA should be considered.In this study, we have described the construction and the use of a series of OriC-plasmids have been successfully used in a number of Mollicutes19. Here, the origin of replication region of M. hyorhinis genome has been identified and was used to construct oriC-plasmids for this bacterium for the first time. The prediction of large oriC (LoriC) region of M. hyorhinis chromosome has identified two dnaA boxes upstream and downstream the dnaA, and this LoriC was proved to be functional in the pGEMT-LoriC plasmid. In the M. hyorhinis transformants, the pGEMT-LoriC plasmid was found to replicate as a free extra-chromosomally before integrating into the host chromosome during passaging, similarly to the pBOT1 plasmid in Spiroplasma citri26.oriC sequences has been shown to influence the recombination efficiency. Furthermore, the presence of a complete dnaA is not essential to driving the replication in oriC-plasmids as in Spiroplasma citri19. Therefore, a plasmid containing a minimum oriC fragment was constructed to decrease the frequency of plasmid integration into the oriC-region of M. hyorhinis15. Indeed, free extra-chromosomal oriC-plasmids were obtained when the pGEMT-MoriC plasmid containing a minimum oriC-fragment (876-bp) used, similar to previous observations19.The length of the M. hyorhinis strain HUB-1 stored in our laboratory is tetracycline sensitive27. The oriC-based plasmids described here have several useful features that will provide flexibility in future genetic studies. The multiple cloning sites located upstream and downstream of tetM and the spiralin gene promoter make these oriC-plasmids compatible for the subsequent cloning of foreign DNA (such as GFP or other antigens) for many applications in M. hyorhinis. Indeed, this may encourage in vivo imaging following M. hyorhinis infection or even development of recombinantly engineered vaccines when foreign antigens are expressed.Tetracycline was chosen as the selection marker as it has been used in multiple mycoplasma species and that hlyC by using our oriC-plasmids, and the data show that, the hlyC is responsible for the hemolytic activity observed in M. hyorhinis. Analysis of the hlyC DNA sequence present in different strains of M. hyorhinis  by the blast and Clustal V methods, has revealed only one base different (in DBS 1050 and GDL-1 strains) in the hlyC sequence  was kindly provided by Professor Xiao Shaobo  and cultured in a modified Friis medium (KM2 medium) containing 20% (v/v) swine serum29 at 37 °C. The solid medium (KM2-Agar) was prepared by adding 0.7% Agar  to the liquid KM2 medium and was incubated at 37 °C until the growth of the visible colonies. For the growth of mutants, tetracycline hydrochloride (Sigma-Aldrich) was added at 5.0 μg/ml and 2.5 µg/ml to the solid and liquid media respectively.The pGEM®-T vector to construct the oriC-plasmids. Images of PCR ethidium bromide-stained agarose gels were acquired and with Gel Doc™ XR + System with Image Lab™ Software . The tetracycline gene, tetM (ID: AGI19285.1) was PCR amplified  and cloned into pGEM®-T vector at SpeI/PstI restriction enzymes sites by infusion cloning methods to generate pGEMT-tetM plasmid. Since common bacterial cloning vectors such as pGEM®-T vector or genes from E. coli cannot express in the M. hyorhinis from their own promoter, the spiralin gene promoter  of M. hyorinis was identified , the flanking regions of dnaA that contains the AT-rich, were PCR amplified with P4A primers, Table dnaA, 563-bp) and with P4B primers, Table dnaA, 313-bp). These two fragments were joined by overlapping PCR  because we hypothesized that if hlyC confers hemolytic activity, it may be easy to measure the hemolytic phenotype. To construct plasmid targets hlyC, both hemolysin left and right arms were at least 450-bp to provide adequate homologous sequences to facilitate recombination at the target gene22. These arms were PCR amplified and cloned to flank tetM as well as the spiralin gene promoter since we aimed to knock-in a functional tetM at the hemolysin site. The left arm (LA) of hlyC  of hlyC  and was used to inactivate hlyC by homologous recombination.We chose to disrupt recA) of E. coli was found to enhance the homologues recombination in M. mycoides subsp. capri7. Therefore, the recA (GI:446885887) was PCR amplified from E. coli BL-21 DNA using P7 primers, Table P8 primers, Table recA  and was also evaluated for its functionality to inactivate hlyC by homologous recombination. All cloning steps were verified by colony PCR and analysis of DNA sequencing. The diagram of the vectors constructions was shown in  and resuspended in 100 µl ice-cold electroporation buffer. Aliquot of 0.1 ml of competent cells was mixed with 20 µg plasmid DNA. The mixture was then placed in a pre-chilled sterile electroporation cuvette  and pulsed immediately with ECM 630 Electroporation System  at 2.5 KV, 125 Ω, and 25 µF. The mixture was immediately diluted with 900 µl of cold KM2 broth, incubated on ice for 5 min, and recovered at 37 °C for 3 hours. The cells were diluted 1:10 and selected on KM2-Agar containing 2.5 µg/ml tetracycline and grown at 37 °C till colonies appeared. Tetracycline-resistant colonies were sub-cultured in 2 ml of KM2-broth containing 5.0 μg/ml tetracycline. Transformants were passaged by inoculating 100 μl of culture into 900 μl of broth containing 5.0 μg/ml of tetracycline.pGEMT-LoriC and pGEMT-MoriC plasmids, the cells were diluted and plated on KM-Agar containing 2.5 µg/ml tetracycline. Individual colonies of M. hyorhinis were sub-cultured in KM2 medium containing 5.0 µg/ml tetracycline at 37 °C several times to eliminate the residual plasmid from the medium. To detect the presence of the plasmids in the transformants, total DNA was extracted from 2 ml M. hyorhinis cultures using a genomic DNA purification kit . We performed a PCR amplifying a fragment of the tetM (part of the oriC-plasmid backbone) using universal tetM screening primers  using SPSS . Supplementary Information


SERPINA1 gene, which encodes alpha-1 antitrypsin . An important function of A1-PI in the lung is to inhibit neutrophil elastase, one of various proteolytic enzymes released by activated neutrophils during inflammation. Absence or deficiency of A1-PI leads to an imbalance between elastase and anti-elastase activity, which results in progressive, irreversible destruction of lung tissue, and ultimately the development of chronic obstructive pulmonary disease with early-onset emphysema. AATD is under-diagnosed, patients can experience long delays before obtaining an accurate diagnosis, and the consequences of delayed diagnosis or misdiagnosis can be severe. Currently, A1-PI therapy is the only available treatment that addresses disease etiology in patients with AATD; however, demonstrating clinical efficacy of A1-PI therapy is challenging. In order to show therapeutic efficacy with traditional endpoints such as forced expiratory volume in one second and mortality, large sample sizes and longer duration trials are required. However, AATD is a rare, slow progressive disease, which can take decades to manifest clinically and recruiting sufficient numbers of patients into prolonged placebo-controlled trials remains a significant obstacle. Despite this, the Randomized, placebo-controlled trial of augmentation therapy in Alpha 1-Proteinase Inhibitor Deficiency (RAPID) and RAPID Extension trial, the largest clinical program completed to date, utilized quantitative chest computed tomography as a sensitive and specific measure of the extent of emphysema. Findings from the RAPID/RAPID Extension program definitively confirmed the benefits of A1-PI therapy in slowing disease progression and provided evidence of a disease-modifying effect of A1-PI therapy in patients with AATD. These findings suggest that the early introduction of treatment in patients with severe emphysema-related AATD may delay the time to death, lung transplantation or crippling respiratory complaints. In addition, there is now limited evidence that A1-PI therapy provides a gain of more than five life-years, supporting previous observations based on registry data. With the clinical efficacy of A1-PI therapy now demonstrated, further studies are required to assess long-term outcomes.Alpha-1 antitrypsin deficiency (AATD) is a common hereditary disorder caused by mutations in the  SERPINA1 gene, which encodes alpha-1 antitrypsin  [1-PI [1-PI in the lung is to inhibit neutrophil elastase, one of various proteolytic enzymes released by activated neutrophils during inflammation. Therefore, absence or deficiency of A1-PI leads to an imbalance between elastase and anti-elastase activity. This results in progressive, irreversible destruction of lung tissue, and ultimately the development of chronic obstructive pulmonary disease (COPD) with early-onset emphysema, which increases the risk of crippling respiratory complaints and premature death [Alpha-1 antitrypsin deficiency (AATD) is a common hereditary disorder caused by mutations in the I) [1-PI . In addiI) [1-PI . An impore death .1-PI therapy. In addition, patients with AATD are often misdiagnosed. Respiratory symptoms, including coughing, wheezing, dyspnea and excessive sputum production [Between 2007 and 2010, the number of at-risk individuals in the USA who were tested for AATD doubled . Despiteoduction .1) below 20% predicted have a 2-year mortality of 40% [The consequences of delayed diagnosis or misdiagnosis can be severe. In addition to lung complications, AATD is associated with liver damage; clinical presentation is variable but ranges from neonatal hepatitis to liver cirrhosis and hepatocellular carcinoma in adults . Currenty of 40% . Betweeny of 40% . In May y of 40% , and fory of 40% .Other less common clinical manifestations of AATD include: panniculitis, a serious skin condition ; and air1-PI therapy is the only available treatment that addresses disease etiology in patients with AATD. Historical registry data suggested a mortality benefit of A1-PI, in addition to a benefit on FEV1 decline in a subgroup of patients with moderate-to-severe baseline FEV1 impairment [1 among patients receiving A1-PI therapy [1-PI therapy in slowing disease progression [Currently, Apairment . Further therapy . However therapy , 20. Morgression , 22.1-PI therapy. In addition, an overview of the RAPID/RAPID Extension program will be provided and relevant post-hoc findings from will be examined. Data were sourced from a literature review conducted on MEDLINE with no language or time period settings applied. Search terms included US National Library of Medicine Medical Subject Headings (MeSH) such as ‘alpha-1 antitrypsin deficiency’ and ‘alpha-1 antitrypsin’ and ‘mortality’. In addition, the reference lists of sourced articles were checked, and the author’s personal literature collection was utilized.This narrative review discusses the relevance of outcome measures to assess AATD-related emphysema progression, and mortality data from previous studies of ACOPD is a progressive condition characterized by airflow obstruction that is not fully reversible . Patholo1 has been traditionally regarded as the ‘gold standard’ endpoint for the assessment and monitoring of obstructive airways disease, and has been proposed as a potential surrogate marker for emphysema progression. However, FEV1 lacks the sensitivity to effectively chart disease progression in patients with AATD. Changes in FEV1 occur slowly and unevenly over time. Therefore, demonstrating a therapeutic benefit would require a placebo-controlled trial of at least 5 years duration, and the recruitment of a minimum of 1000 subjects [1 is in the normal range [The early identification of these pathological changes is key to preventing widespread destruction of the lung parenchyma. FEVsubjects . Comparesubjects , the recal range , 27.1 has been used to stratify disease severity in COPD in general, and mortality risk in patients with AATD in particular. Findings of an early National Heart, Lung, and Blood Institute (NHLBI) registry study of patients with severe AATD (n = 1129) showed that, compared with patients not receiving active treatment, the use of A1-PI therapy was associated with lower mortality  [1 % predicted below 50%. More recent analysis of these data further stratified patients based on their baseline FEV1 levels and other lung function parameters. A1-PI therapy was shown to be associated with improved survival in patients with baseline FEV1 % predicted <20% (n = 161), <30% (n = 424) and 30–65% ; a benefit that was not observed in patients with baseline FEV1 % predicted >65%  [1 of 35–49% predicted would need to be followed in a 5-year study to detect a 40% reduction in mortality [Despite its limitations, FEV = 0.02) . This be = 0.38) . NeverthTD n = 11 showed t1-PI products on emphysema disease progression, and permitted its use as a primary endpoint in Phase IV studies [1, the decline in PD15 is linear, which makes it a more consistent measure across the broad spectrum of disease severity seen in patients with AATD [In 2009, the US Food and Drug Administration (FDA) approved computed tomography (CT) as an appropriate clinically meaningful endpoint to assess the efficacy of therapy with intravenous A studies . This ap studies . CT dens studies –33. Percith AATD .1 decline [r = 0.338 for FEV1 % predicted , indicating that change in physiological measurements varies with their baseline values, whereas the change in PD15 does not. A 30 month longitudinal study across five centers indicates that changes in lung density, FEV1 and DLCO may also occur independently of the levels measured at baseline [The rate of decline in CT lung density has been shown to correlate with FEV decline –22, 31. al aging . However1, for predicting mortality in patients with AATD [1-PI therapy [In line with its greater sensitivity as a measure of emphysema progression, CT scanning has also been shown to be superior to lung function parameters, especially FEVith AATD . Furtherith AATD . Howeverith AATD . To date therapy –22.1-PI therapy may reduce the incidence and severity of exacerbations. [post hoc analysis of data from the EXAcerbations and Computed Tomography scan as Lung End-points (EXACTLE) trial indicated that although augmentation therapy was associated with a reduction in exacerbation severity, it did not alter exacerbation frequency [Studies have also evaluated clinical- and patient-reported outcome measures to assess therapeutic efficacy, including health status and mortality. It is well established that AATD-related emphysema impacts on a patient’s quality of life (QoL), and that this occurs at a younger age than non-AATD-related COPD , 37. Stubations. , 41. A prequency . However1-PI therapy for the treatment of AATD-associated emphysema was first demonstrated in the 1980s [1-PI was shown to increase plasma levels above the protective threshold level (>11 μM) and restore the protease-antiprotease balance within the lung. However, the Danish-Dutch [1-PI therapy to prevent the progression of emphysema in patients with AATD. Although neither trial reported a significant effect of A1-PI therapy on pulmonary function, CT analysis showed a trend towards a more favorable effect of treatment, which was suggestive of lung tissue preservation compared with placebo. Furthermore, a combined analysis of both trials confirmed a significant reduction in the decline in lung density with A1-PI therapy [The feasibility of Ahe 1980s , 43. Infsh-Dutch  trials w therapy .1-PI therapy in patients with AATD completed to date, is the only trial specifically designed to assess the disease-modifying effect of treatment. [1-PI therapy compared with placebo ; corresponding to a 34% reduction in the progression of emphysema in favor of A1-PI therapy [1-PI therapy, as evaluated by an FDA endorsed endpoint, was demonstrated [The RAPID/RAPID Extension program, in addition to being the largest trial of Aeatment.  The prog therapy , 22. Fornstrated .1-PI therapy. Overall, from Day 1 to Month 48, treatment differences were shown to favor the Early-Start group , substantiating continued efficacy over 4 years. During RAPID-OLE (Month 24 to Month 48), the rate of decline in lung density was similar between the Early-Start and Delayed-Start groups  [p = 0.001) [1-PI therapy was not regained with the initiation of active treatment [1-PI therapy in patients with AATD.This beneficial effect was confirmed in RAPID-OLE in which all patients received APID-RCT) . Further= 0.001) . Howeverreatment . Taken t1 [Overall, between Day 1 and Month 48, no significant differences were observed between treatment groups in secondary endpoints such as quality of life (St George’s Respiratory Questionnaire score) or pulmonary function tests, including FEV1 , 22. The1 % predicted of 48.5%, the rate of decline in lung density was variable with a mean decline of −1.13 g/L/year [1 % predicted of 50.2%, the annual rate of lung density decline was −1.31 g/L/year [The rates of decline in lung density in patients with AATD as seen in the RAPID/RAPID Extension program are higher than those reported in other trials of lung-related conditions. In patients with COPD, and a mean baseline post-bronchodilator FEVg/L/year . The decg/L/year .post-hoc analysis of the RAPID/RAPID Extension program investigated the time to respiratory crisis for progressive emphysema, defined as death, lung transplant or a crippling respiratory condition. Seven patients withdrew from the 4-year program  due to a respiratory crisis, with an average final recorded lung density of approximately 20.5 g/L (Table 1-PI-treated patients (−1.51 g/L/year) with placebo-treated patients (−2.26 g/L/year), the projected gain in life-years  was approximately 5.6 years , and is only a preliminary estimate. In addition, the analysis presumes a linear decline in loss of lung density. Furthermore, whilst the analysis aimed to explore a mortality benefit, the trials in the RAPID program were not sufficiently powered to assess mortality [n = 1129), would be needed to refine and validate this approach. Three deaths were recorded in the placebo arm of RAPID-RCT compared with one death in the active treatment arm over the first two years. An additional death was reported in the Early-Start group of the 2-year RAPID-OLE.This ortality ; a subst1-PI therapy is greater than the difference between the whole-lung densities of those who died and those who survived [p = 0.052) [However, the life-years gained prediction is supported by historical survival improvement observations based on NHLBI Registry data . In addisurvived . The autsurvived . Even cl= 0.052) .1-PI therapy. It has also been suggested that a reduction in the progression of emphysema with A1-PI therapy may reduce the deterioration in health status [1-PI therapy in order to minimize and/or delay the onset of the deleterious health consequences associated with AATD.Evidence is beginning to indicate an improvement in survival in patients with AATD receiving Ah status . In the 1-PI therapy may pose a considerable burden on patients, their lifestyle, their families, and the healthcare system. Depending on healthcare structure, weekly intravenous administration of A1-PI may require a long journey to the hospital or the treating physician responsible for administering the treatment. This inconvenience could be addressed in part by home-treatment, whereby the infusion is administered by a visiting nurse [However, as with other chronic illnesses, Ang nurse  or the png nurse . Anotherng nurse . Extendeng nurse .1-PI is currently available. While intermittent shortage of drug supply was an issue in the late 1990s and early 2000s, the increasing number of companies with licensed A1-PI products has helped to eliminate this problem. However, as with many treatments requiring plasma products or solely produced for the treatment of rare diseases it comes at a cost.In contrast to hemophilia therapies, where plasma-derived products have in part been replaced by recombinant products, no recombinant A1-PI therapy has the potential to offer benefits with the life-years extension in comparison with the high mortality and morbidity rates associated with lung transplantation. Between January 1994 and June 2011, adults who underwent either a single of double lung transplantation , which included patients with COPD (34%) and AATD (6%), had a median survival of 5.6 years [p = 0.006) [1-PI therapy.In the long-term, A.6 years . Surviva.6 years . In addi.6 years . In pati= 0.006) . HoweverFurther study and validation of the terminal lung density threshold used in the life-years gained analysis is required. It would allow better planning for transplantation in patients with AATD, and is also important in terms of disease prognosis and life planning, and perhaps even the decision to treat.1-PI therapy is challenging. In order to show therapeutic efficacy with traditional endpoints such as FEV1 and mortality, large sample sizes and longer duration trials are required. However, AATD is a rare, slow progressive disease, which can take decades to manifest clinically [1-PI therapy in patients with AATD [Demonstrating clinical efficacy of Ainically , and recith AATD , 22, wit